Inflammatory events in the central nervous system (CNS) contribute to the disease process in Multiple Sclerosis (MS), Alzheimer's Disease (AD), and Spinal Cord Injury (SCI), and activated macrophages/microglia are central to this response. Immunological activation of these cells leads to the production of a wide array of cytokines, chemokines, matrix metalloproteinases and neurotoxins, and ultimately to glial/neuronal injury and death. The CD40 molecule has an important role in promoting inflammatory responses by macrophages/microglia, since interaction with its cognate ligand, CD 154, leads to secretion of cytokines and neurotoxins. Thus, expression of CD40 on macrophages/microglia in MS and AD brain, and in the injured spinal cord, may be a key determinant of the complex neuroimmunologic cascades that typify these diseases/injury. We hypothesize that aberrant CD40 expression by macrophages/microglia, induced by cytokines such as IFN-gamma and TNF-alpha, contributes to inflammatory responses in the CNS. We also propose that strategies to suppress CD40 expression will attenuate inflammation and neuronal damage within the CNS, which will ultimately be of benefit in MS, AD and SCI. The mediators that regulate expression of CD40 in macrophages/microglia (both induction and inhibition) function at the level of gene transcription, thus it is imperative that we gain a better understanding of the molecular mechanisms involved in these responses. We will elucidate the contribution of the TNF-alpha signaling pathway and subsequent NF-kappaB activation to IFN-gamma induced CD40 gene expression in macrophages/microglia (Aim 1). Next, we will determine the interactions between STAT-1alpha and NF-kappaB transcription factors and the CD40 promoter, and between transcription factors and various co-transactivators including CBP, p300 and CARM1, to fully understand CD40 gene expression (Aim 2). Immunoregulatory cytokines such as IL-4 and IL-10, and neurotrophins such as NGF, inhibit CD40 expression in macrophages/microglia. In Aim 3, we will determine the molecular mechanism(s) underlying suppression of CD40 expression in these cells. Lastly, we will analyze the expression and function of CD40 and CD154 in vivo, utilizing SCI as a paradigm of neuroinflammation (Aim 4). We will assess CD40 and CD154 expression after SCI, the cell type(s) expressing these proteins and the functional importance of CD40-CD154 interactions in SCI utilizing CD40 and CD154-deficient mice. Our proposed studies will provide a comprehensive assessment of CD40 production and function in macrophages/microglia, thereby setting the foundation for future therapeutic manipulation of this critical immunoregulatory protein.